The present invention disclosed herein relates to complexes of the Hermansky-Pudlak Syndrome (HPS) protein with other proteins including, but not limited to, complexes of HPS with: 14-3-3 eta, Hrs, BMK1 alpha kinase, CDK2, Nuclear factor NF90, Atrophin-1, DGS-1, HPIP1 and human HN1 homolog protein. The present invention further relates to antibodies specific for HPS complexes, and their use in, inter alia, screening, diagnosis, prognosis and therapy. The present invention further relates to the HPIP1 and human HN1 homolog protein nucleic acid, protein and derivatives, fragments and analogs thereof.
Hermansky-Pudlak syndrome (HPS) is a genetic disorder characterized by defective lysosome-related organelles. Although the frequency of HPS is quite low in the general population, the frequency is markedly higher in certain genetically-isolated population groups. For example, HPS occurs in northwest Puerto Rico with a prevalence of 1 in 1800. In humans, HPS is characterized by the symptomatic triad of oculocutaneous albinsim, platelet dysfunction (i.e., mild to moderate bleeding diathesis), and ceroid deposition. Tissue accumulation of ceroid pigment (ceroid desposition) is considered to cause several serious complications, including progressive pulmonary fibrosis leading to death in the fourth or fifth decades. The primary defect in HPS involves affects on the contents and/or the secretion of several subcellular organelles, including lysosomes, melanosomes, and platelet-dense granules. See e.g., Swank, et al., 1998. Pigment Cell Res. 11:60-80; Erickson, 1997. Proc. Natl. Acad. Sci. USA 94:8924-8925. The synthesis of pigmented melanosomes is compromised in HPS patients, wherein functional melanocytes are quantitatively reduced in number and/or are qualitatively abnormal, thus resulting in albinism of both dermal and keratinized tissues (e.g., the skin and hair). See e.g., Gardner, et al., 1997. Proc. Natl. Acad. Sci. USA 94:9238-9243. The prolonged bleeding time associated with HPS is due to the lack of the storage organelles, platelet-dense granules, which are required for ADP-release and platelet aggregation. See e.g., Holmsen, et al., 1979. Ann. Rev. Med. 30:119-134. The pulmonary fibrosis and granulomatous colitis demonstrated in some cases of HPS is due to the accumulation of ceroid lipofuscin in lysosomes of reticuloendothelial cells, bone marrow and lung macrophages, gastrointestinal mucosal cell, and other cell types. See e.g., Harmon, et al., 1994. J. Lab. Clin. Med. 123:617-627.
Similar physiological abnormalities have been shown to occur in the pale ear (ep) mouse. See Lane and Green, 1967. J. Heredity 58:17-20. Homozygous recessive ep mice have decreased skin and eye pigment at birth, as well as abnormal organelle function resulting in an increase of serum lysosomal enzymes. See Novak and Swank, 1979. Genetics 92:189-204. Similar deleterious physiological symptomology was also described in a murine model of Chediak-Higashi Syndrome (CHS), within the beige mouse. CHS is, like HPS, an autosomal recessive genetic disorder. Symptoms in affected patients involve hypopigmentation, immunological deficiency, a bleeding tendency and a neurological disorder, probably due to protein sorting defects, especially in secretory lysosomes of granular cells. See Spritz, 1998, J. Clin. Immunol. 18:97-105. Chediak-Higashi syndrome proteins (LYST and LYST-2) have been identified in mouse and in man. LYST interacting proteins include 14-3-3 protein, HS1 (14-3-3 beta) protein, Hrs, BMK1 alpha kinase, KB07, Efs, OS9, casein kinase II beta SU, calmodulin, Troponin, Importin beta, Fte-1, estrogen-receptor related protein, Imogen 38, Atrophin-1, GBDR1, DGS-I, noHPSin (KIAA0607), OPA containing protein, M4 protein, LIP1 (Tcp-10 homolog), LIP2 (L17 homolog), LIP3 (Roaz protein homolog), LIP4 (hnRNP-e2 homolog), LIP5, LIP6 (Ns2-3 homolog), LIP7 (TCP10A homolog), LIP8 (KAP4L homolog), LIP9 (etr-1 homolog), and LIP10, as described in U.S. patent application Ser. No. 09/054,956.
The gene encoding a HPS-related protein has recently been cloned in both humans and mice. See Oh, et al., 1996. Nat. Genet. 14:300-306; Gardner, et al., 1997. Proc. Natl. Acad. Sci. USA 94:9238-9243. Sequence analysis has predicted this polypeptide (hereinafter the xe2x80x9cHPS proteinxe2x80x9d) to be a component of cytoplasmic organelles due to the presence of a putative transmembranal region. The mRNA encoded by the HPS gene in ep mice appears to be very widely expressed.
In summary, it is most likely that both human and murine HPS symptoms results from a defect in a protein which is required for the normal assembly, maturation, and/or structure of numerous, diverse subcellular organelles. Accordingly, HPS is likely to be centrally implicated in pathological processes, including but not limited to, oculocutaneous albinism, fibrotic lung disease, and various clotting, bleeding, and neurodegenerative disorders.
As previously discussed, HPS patients have been reported to suffer from several serious medical conditions, including oculocutaneous albinsim, a bleeding diathesis, and ceroid deposition, often accompanied by severe fibrotic lung disease and granulomatous colitis. However, despite the recognition of these HPS-associated syndromes, no curative therapeutic intervention currently exists for this disease. Currently, only symptomatic treatment can be offered. One potential area of difficulty involves a lack of understanding regarding the interaction of the HPS protein with other cellular proteins. The elucidation of these potential interactions may provide the means for the subsequent development of an diagnostic assay and/or a therapeutic modality for HPS and its associated diseases.
The following sections will discuss the various proteins which have been shown to interact with the HPS protein. These HPS protein-interacting-proteins hereinafter xe2x80x9cHPS-IPxe2x80x9d may be differentiated into proteins which are involved in signaling processes and protein trafficking (14-3-3 eta, Hrs, BMK1 alpha, CDK2, NF90), those proteins involved in neurodegenerative and developmental disorders (Atrophin I, DGS-1) and those previously-uncharacterized, novel proteins (HPIP1, HN1 homolog). Interestingly, five of these nine interacting proteins, or similar proteins, were found to interact as well with the LYST protein, as described by Nandabalan and Kingsmore in U.S. patent application Ser. No. 09/054,956 (Apr. 3, 1998): 14-3-3 protein, HS1 (14-3-3 beta) protein, Hrs, BMK1 alpha kinase, Atrophin-1, and DGS-I. CDK2, NF 90, HPIP1, and HN1 homolog were not found to interact with LYST. Table I provides an overview of all HPS interacting proteins and their interacting domains as disclosed in by the present invention.
It should be noted that the citation of a reference in this or in any other section of the specification should not be construed as an admission that such reference is prior art to the present invention disclosed herein.
(A) Proteins Involved Signaling Processes and Protein Trafficking
(i) 14-3-3 eta
The caHPSoxy-terminal region (starting at nucleotide 764) of the 14-3-3 protein eta isoform (GenBank Accession Number X80536; Ichimura-Ohshima, et al., 1992. J. Neurosci. Res. 31:600-605) was found to interact with HPS protein in this invention. The nucleotide and amino acid sequences of a Hermansky-Pudlak Syndrome Protein sequence provided in GenBank Accession Number U65676 are as follows:
Interestingly, the highly homologous proteins 14-3-3 protein and HS1 (14-3-3 beta) protein were described as LYST interacting protein in U.S. patent application Ser. No. 09/054,956 (filed Apr. 3, 1998). The highly conserved 14-3-3 family of proteins is found in a broad range of organisms and tissues and have been associated with many diverse biological functions, including signal transduction, exocytosis and cell-cycle regulation. The 14-3-3 proteins have been demonstrated to associate with a wide-range of cellular and viral polypeptides involved in signal transduction, cell cycle regulation and/or oncogenesis, suggesting that they participate in cellular growth regulation. See e.g., Aitken, 1995. Trends Biochem. Sci. 20:95-97. For example, the eta isoform interacts with several kinases, implicating 14-3-3 eta proteins in intracellular signal transduction cascades and cellular protein networks. The nucleotide and amino acid sequences of a 14-3-3 eta sequence provided in GenBank Accession Number X80536 is as follows:
In addition, calcium-dependent exocytosis in permeabilized adrenal chromaffin cells has been demonstrated to be mediated by several proteins, including, but not limited to, 14-3-3 proteins (see e.g., Morgan and Burgoyne, 1992. Nature 355:833-836); alpha-SNAP proteins (see e.g., Morgan and Burgoyne, 1995. EMBO J. 14:323-239); and protein kinase C (see e.g., Morgan and Burgoyne, 1992. Nature 355:833-836). Furthermore, 14-3-3 proteins may enhance catecholamine release in permeabilized cells by reorganizing the cortical actin-barrier to allow the increased availability of secretory vesicles for exocytotic release. See e.g., Roth and Burgoyne, 1995. FEBS Letters 374:77-81.
The 14-3-3 family of proteins (including the eta isoform), activate tryptophan and tryptophan hydroxylase in brain tissue, which is one of the rate-limiting steps in catechol amine and serotonin neurotransmitter biosynthesis (see e.g., Banik, et al., 1997. J. Biol. Chem. 272:26219-26225). 14-3-3 proteins have been demonstrated within the neurofibrillary tangles seen in Alzheimer""s Disease. This association may be due to the 14-3-3 protein""s affect on MAP kinase signaling, which causes hyper-phosphorylation of the tau protein. This tau protein hyper-phosphorylation is believed to lead to the formation of the paired helical filaments seen in the brains of Alzheimer""s Disease victims. See e.g., Layfield, et al., 1996. Neurosci. Lett. 209:57-60. Moreover, 14-3-3 proteins have also been shown in the cerebrospinal fluid of patients with Creutzfeldt-Jakob disease. See e.g., Rosenmann, et al., 1997. Neurol. 49:593-595. In summary, there is a strong implication for the involvement of the 14-3-3 family of proteins, including the eta isoform, in neurodegenerative disorders. In addition, 14-3-3 eta plays roles in is signal tranduction, cell cycle regulation and oncogenesis.
(ii) Hrs Protein
Another protein which was found to interact with the HPS protein was Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) protein [GenBank Accession No. D84064; Lu et al., 1998. Gene 213:125-135]. In addition, Hrs was described as interactant of the Chediak-Higashi Protein LYST (see U.S. patent application Ser. No. 09/054,956, filed Apr. 3, 1998). Hrs is a 115 Kdal cytoplasmic protein with a structurally-conserved, putative zinc-finger binding domain and several proline-rich regions. See e.g., Komada and Kitamura, 1995. Mol. Cell. Biol. 15:6213-6221. The phosphorylation of tyrosine residues within the Hrs protein may be induced by the treatment of cells with epidermal growth factor or platelet-derived growth factor. It is thus likely that Hrs plays a role in the intracellular signaling pathway of these aforementioned growth factors. The nucleotide and amino acid sequences of a Hrs sequence provided in GenBank Accession Number D84064 is as follows:
Hrs has been shown to exhibit an 80% homology with rat Hrs-2, an enzyme with ATPase catalytic activity. Hrs-2 was characterized as a brain protein which interacts with SNAP-25, a plasma membrane protein involved in vesicular transport (i.e., vesicular docking and fusion). Synaptic vesicle docking and calcium-dependent exocytosis require the specific interaction of various synaptic vesicle membrane proteins (e.g., VAMP and synaptogamin) with their plasma membrane-localized counterparts (e.g., SNAP-25 and syntaxin). See Sollner, et al., 1993. Cell 75:409-418. It was then demonstrated that Hrs-2 functioned to significantly inhibit secretion in a dose-dependent manner, and thus, may be a modulator of exocytosis. Specifically, the binding of Hrs-2 to SNAP-25 is inhibited by calcium at a concentration which is required to support synaptic transmission. Thus, Hrs-2 (and the homologous human protein Hrs) may act as regulators of secretory processes through calcium- and nucleotide-dependent modulation of vesicle-trafficking protein complexes (e.g., SNAP-25). See e.g., Bean, et al., 1997 Nature 385:826-829.
(iii) BMK1 alpha kinase
The caHPSoxy-terminal region (starting at nucleotide 2431) of the BMK1 alpha kinase (GenBank Accession Number U29725) was found to interact with HPS protein, as disclosed in this ivention. Interestingly, the same domain of BMK1 alpha kinase was also found to interact with the LYST protein that plays a key role in Chediak Higashi Syndrome. The activated protein kinase BMK1 is part of a distinct signalling pathway that is required for proliferation and progression through the cell cycle (see, e.g., Lee, et al., 1995. Biochem. Biophys. Res. Commun. 213(2):715-724. The nucleotide and amino acid sequences of a BMPK1 kinase sequence provided in GenBank Accession Number U29725 is as follows:
(iv) CDK2
Another HPS protein-IP is the cell cycle regulator CDK2 (GenBank Accession Number X61622; Elledge and Spottswood, 1991. EMBO J. 10:2653-2659). In the mammalian cell cycle, the transition from the G1 phase to DNA replication phase is regulated by the cyclin-dependent kinases (CDKs). Activities of CDKs are controlled by association with cyclins and reversible phosphorylation reactions. An additional level of regulation is provided by inhibitors of CDKs. CDK2 is expressed in, for example, but not limited to, the majority of squamous cell carcinomas, small cell carcinomas, and large cell carcinomas. Higher CDK2 kinase activity is critical for promoting cell cycle progression and unrestrained proliferation of tumor cells. The nucleotide and amino acid sequences of a CDK2 sequence provided in GenBank Accession Number X61622 is as follows:
(v) Nuclear Factor NF-90
The caHPSoxy-terminal region (starting at nucleotide 1930) of nuclear factor NF90 [GenBank Accession Number U10324; See e.g., Kao, et al., 1994. J. Biol. Chem. 269:20691-20699) was found to interact with the HPS protein, as disclosed in this invention. The nuclear factor of activated T cells (NFAT) has been shown to regulate gene expression of the lymphokine Interleukin-2 (IL-2) which is secreted following T-cell activation. See e.g., Marcoulato, et al., 1998. J. Interferon Cytokine Res. 18:351-355. The 90 and 45 Kdal subunits (i.e., NF90 and NF45) of NFAT specifically bind to the antigen receptor response element of the IL-2 promoter. NFAT is the nuclear-target of both T-cell stimulation signals and the immunosuppressant activity of the drugs cyclosporine and FK-506; whereas NF90 and NF45 are substrates for DNA-dependent protein kinase (DNA-PK) in vitro. In addition, recombinant NF90 has been found to promote the formation of a complex between the subunits of DNA-PK and DNA. See e.g., Lu, et al., 1998. J. Biol. Chem. 273:2136-2145. The nucleotide and amino acid sequences of a Nuclear Factor NF90 sequence provided in GenBank Accession Number U10324 is as follows:
(B) Neurodegenerative and Developmental Disorder Proteins
(i) Atroiphin-I
The caHPSoxy-terminal region (starting at nucleotide 2649) of atrophin-I (GenBank Accession Number U23851; Margolis, et al., 1996. Brain Res. Mol. Brain Res. 36:219-226) was found to interact with HPS protein, as disclosed in this invention. Interestingly, the same domain of atrophin-I was also found to interact with the LYST protein. Although the exact function of atrophin-1 is unknown, atrophin-I is the protein encoded by the gene involved in dentatorubral pallidoluysian atrophy (DRPLA; Smith""s Disease), a rare, progressive and fatal autosomal dominant neurological disorder. DRPLA is characterized by neuronal degeneration, especially in the cerebellar dentate nucleus. Clinical symptomology include variable combinations of myoclonus epilepsy, cerebellar ataxia, choreoathetosis and dementia. DRPLA has been shown to result from the expansion of a CAG trinucleotide repeat encoding the amino acid glutamine. The DRPLA gene product has been primarily localized within the neuronal cytoplasm by in situ hybridization (see e.g., Yazawa, et al., 1995. Nat. Genet. 10:99-103) and it is wide-spread throughout the cerebral and cerebellar regions (see e.g., Knight, et al., 1997. J. Neurol. Sci. 146:19-26). The nucleotide and amino acid sequences of a Atrophin-I sequence provided in GenBank Accession Number U23851 is as follows:
Atrophin-1 interacting proteins (AIPs) containing multiple WW domains were identified (see, e.g., Wood et al, 1998, Mol. Cell. Neurosci. 11:149-60). Two of these proteins are multidomain proteins containing a number of protein-protein interaction modules. The other three AIPs are highly homologous, each having four WW domains and a HECT domain characteristic of ubiquitin ligases.
(ii) DiGeorge Syndrome (DGS)-I Protein
A further HPS protein-IP disclosed herein is the DiGeorge Syndrome (DGS)-I protein (GenBank Accession Number L77566; see e.g., Gong, et al., 1996. Hum. Mol. Genet. 5:789-800). DGS-I was also identified as LYST interactant (see U.S. patent application Ser. No. 09/054,956; filed Apr. 3, 1998). One in 4,000 children is born with chromosome 22 deletion syndrome (DiGeorge syndrome), making it one of the most common genetic abnormalities in children. Patients with DiGeorge Syndrome possess deletions of the chromosomal region 22q11.2. The DGS-I gene has been localized to a DGS-critical region of chromosome 22 which encodes for protein consisting of 476 amino acid residues. Clinical symptomology associated with DGS include cardiac defects, thymic hypoplasia cardiac defects, abnormal facial features, immune deficiencies, cleft palate and low blood calcium.
(C) HPS Protein-IPs Encoded by Novel Genes
(i) HPIP1
One heretofore uncharacterized gene, referred to as HPIP1, has thus far been identified by the present invention disclosed herein. However, the translational products of the mRNA of these genes have not been defined and, thus no function has yet been assigned.
(ii) Human HN1 Homolog
Another heretofore uncharacterized human gene (referred to as HN1 homolog) was identified as the human homolog to mouse gene HN-1 (see Tang et al., 1997. Mamm. Genome 8:695-6). Murine Hn1 is expressed in many fetal and adult tissues. The highest levels of expression are found in hemopoietic cells, including day 10 yolk sac, blood islands-derived circulating erythroblasts, day 13 fetal liver, adult bone marrow and spleen. The expression is also very high in day 17 fetal brain, while the expression in adult brain is considerably lower. The nucleotide and amino acid sequences for the human homolog of murine HN-1 are disclosed by the present invention.
The present invention disclosed herein is based upon the novel finding that certain cellular proteins bind to, and form complexes with, the Hermansky-Pudlak Syndrome (HPS) protein. Accordingly, the present invention discloses protein complex compositions which are comprised of the HPS protein bound to (i.e., complexed with) a protein which recognizes and interacts with the HPS protein. It should be noted that a protein which forms a complex with HPS protein hereinafter will be designated an xe2x80x9cHPS protein-IPxe2x80x9d for HPS protein-Interacting Protein; and of the HPS protein and an HPS protein-IP hereinafter will be designated as xe2x80x9cHPS proteinxe2x80xa2HPS protein-P complexes.xe2x80x9d
More specifically, the present invention is directed to complexes of HPS protein and complexes of the derivatives, fragments and/or analogs of HPS protein with HPIP1 and human HN1 homolog, as well as with the derivatives, fragments and/or analogs of these aforementioned HPS protein-IPs.
The present invention further discloses methodologies of screening for proteins which interact with the HPS protein or derivatives, fragments and/or analogs, thereof. Preferably, the method of screening is a yeast two-hybrid assay system, or a variation thereof.
The present invention further discloses the nucleotide and amino acid sequences of human HPIP1 (and homologs of other species) and of human HN1 homolog, as well as derivatives, fragments and analogs thereof. Nucleic acids which are complementary to (i.e., possess the ability to hybridize to), specific nucleotide sequences (e.g., the inverse complement of the foregoing sequences), are also provided. An inverse complement is a nucleic acid sequence which possesses a complementary sequence, running in reverse orientation to the coding strand, such that the inverse complement would hybridize without mismatches to the nucleic acid strand. Thus, for example, where the coding nucleic acid strand is hybridizable to a nucleic acid sequence with no mismatches between the coding strand and the hybridizable strand, then the inverse complement of the hybridizable strand is identical to the coding strand.
The present invention also discloses derivatives, fragments and/or analogs of HPIP1 and human HN1 homolog and which possess biological activity (i.e., they are capable of displaying one or more known functional activities of the wild-type HPIP1 or human HN1 homolog protein. Such biological activities include, but are not limited to: (i) the ability to bind to, or compete for interaction with, the HPS protein; (ii) antigenicity (i.e., the ability to bind to, or compete with, HPIP1 or human HN1 homolog for binding to an anti-HPIP1 or anti-human HN1 homolog antibody, respectively and (iii) immunogenicity (i.e., the ability to generate an antibody which is specific for, and binds to, HPIP1 and human HN1 homolog protein.
Methodologies for the production of the HPS proteinxe2x80xa2HPS protein-IP complex and of the HPIP1 and human HN1 homolog protein, and derivatives and analogs of these individual proteins and/or protein complexes (e.g., by recombinant means), are also disclosed. Pharmaceutical compositions comprising same are also provided herein.
The present invention further discloses methodologies for the modulation (i.e., the inhibition or enhancement) of the activity of HPS proteinxe2x80xa2HPS protein-IP complexes, and methods of modulating the HPIP1 and human HN1 homolog proteins. The individual protein components of these complexes have been implicated in various cellular functions, including but not limited to: physiological processes (e.g., vesicular transport, protein trafficking, pigmentation regulation, and platelet function) and pathological processes (e.g., oculocutaneous albinism, platelet dysfunction, neurodegenerative disease, and fibrotic lung disease).
In accord, the present invention also discloses methodologies for screening specific proteins or protein complexes including, but not limited to: (i) screening for the HPS proteinxe2x80xa2HPS protein-IP complex, the HPIP1 and human HN1 homolog proteins, as well derivatives and analogs of the HPS proteinxe2x80xa2HPS protein-IP complex; (ii) screening for HPIP1 and human HN1 homolog mRNA and (iii) screening the HPIP1 protein and human HN1 homolog for their ability to alter cell functions, particularly those cell functions in which the HPS protein and/or an HPS protein-IP have been implicated.
The present invention further discloses diagnostic and prognostic screening methodologies, as well as therapeutic and prophylactic compositions which are based upon: (i) HPS proteinxe2x80xa2HPS protein-IP complexes (including the nucleic acids encoding the individual proteins which participate in the formation of the complexes) and (ii) the HPIP1 protein and their encoding nucleic acid. Therapeutic compounds of the present invention include, but are not limited to: (i) HPS proteinxe2x80xa2HPS protein-IP complexes where one or both members of said complex is a derivative, fragment or analog of the HPS protein and/or an HPS protein-IP; (ii) HPIP1 protein and human HN1 homolog and derivatives, fragments or analogs thereof; (iii) antibodies to the protein or the derivatives, fragments or analogs thereof and (iv) nucleic acids encoding the aforementioned proteins, or their derivatives, fragments or analogs. Therapeutic compounds may also include the generation of antisense nucleic acids specific for the nucleotide sequences encoding both the HPS proteinxe2x80xa2HPS protein-IP components and the HPIP1 and human HN1 homolog protein. In addition, diagnostic, prognostic and screening kits are also disclosed herein.
Animal models and methodologies related to the screening of modulators (e.g., agonists, antagonists and inhibitors) of the biological activity of an HPS proteinxe2x80xa2HPS protein-IP complex, or of an HPIP1 protein, are also provided in the present invention. Similarly, methodologies related to the identification of molecules which inhibit or, alternatively, increase formation of HPS proteinxe2x80xa2HPS protein-IP complex complexes are also disclosed.